Direct-seq: programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen

Genome Biol. 2020 Jun 8;21(1):136. doi: 10.1186/s13059-020-02044-w.

Abstract

CRISPR-based genome perturbation provides a new avenue to conveniently change DNA sequences, transcription, and epigenetic modifications in genetic screens. However, it remains challenging to assay the complex molecular readouts after perturbation at high resolution and at scale. By introducing an A/G mixed capture sequence into the gRNA scaffold, we demonstrate that gRNA transcripts could be directly reverse transcribed by poly (dT) primer together with the endogenous mRNA, followed by high-content molecular phenotyping in scRNA-seq (Direct-seq). With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Gene Expression Profiling
  • Genotyping Techniques
  • HEK293 Cells
  • Humans
  • K562 Cells
  • RNA, Guide*
  • Sequence Analysis, RNA*
  • Single-Cell Analysis*

Substances

  • RNA, Guide